Name: Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection (Video) | JoVE Journal
Uploaded: 2017-06-13T14:00:00.000+00:00
Duration: 10 min 26 s
Description: Learn to rapidly initiate, extend and connect neurites using poly-D-lysine-coated beads and micropipette guidance in microfluidic chambers. This method enables precise control of neuronal growth for neuroscience research.

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5 minutes

We would like to thank Yoichi Miyahara for many helpful discussions and insights. MA and PG acknowledge funding from NSERC.

All procedures detailed below were approved by McGill University&#39;s Animal Care Committee and conformed to the guidelines of the Canadian Council of Animal Care.
1. Standardization of Neuronal Cultures Using Microfluidic Devices: Device Assembly
<ol>
	<li>Select a suitable microfluidic device for the desired experiment. To connect neurons within the same population, use the Neuro Devices (Figure 1) and to connect neurons in different populations use the Co-Culture Devices (Figure 6).</li>
	<li>Clean and prepare the desired number of sterile coverslips or glass bottom dishes. For best results on plastic surfaces use 35 mm dishes, on glass use 25 mm coverslips, or 35 mm glass bottom dishes. Select glass thickness based on the imaging system, for instance 0.15 mm.</li>
	<li>Coat the dishes or coverslips with 0.5-1 mL of 100 &#181;g/mL PDL for 2 h or overnight at room temperature. 
	Note: Protocol can be paused here and resumed the following day if desired. Furthemore, dishes can be coated with borate buffer-diluted PDL, Poly-L-Lysine (PLL), laminin or any other cell adhesion molecule.</li>
	<li>Wash the dishes twice with water (do not use phosphate-buffered saline (PBS) as salt crystals may block the channels), remove all the liquid and let it dry in a sterile environment such as a biosafety cabinet for 5-10 min or until the surface is completely dry. 
	Note: Be careful to ensure that the coverslips are absolutely dry, as any remaining liquid will interfere with the adherence of the microfluidic systems.</li>
	<li>Place microfluidic devices with patterns facing up under UV light in a sterile environment (biosafety cabinet) for 10 min. Be sure to follow sterile procedures when working in the biosafety cabinet10.</li>
	<li>Using tweezers place a microfluidic device with pattern facing down in contact with the clean coverslip/dish. Use the tweezers to softly press the device so that it adheres to the glass. 
	Note: The transparency of the adhered region will be visible when looking against the light. Make sure all corners are contacting the glass. Do this to all microfluidic devices. See Figure 1a .</li>
	<li>To fill the single population device with medium, point the pipette towards the channels and add 50 &#181;L of complete cell medium supplemented with serum-free B-27 (volume ratio 1:50) and 500 &#181;g/mL penicillin/streptomycin/glutamine (collectively called NBM) to the right upper well, and then add another 50 &#181;L to the well lying diagonally to it. Do this for all devices, making sure that the medium flows between wells. Next, add 50 &#181;L of medium to the remaining 2 wells. See Figure 2a .
	<ol>
		<li>To fill the multiple population device with medium, point the pipette towards the channels and add 30 &#181;L of complete NBM to the right wells, refer to Figure 6a. Do this for all devices, making sure that the medium flows between wells. Next, add 50 &#181;L of medium to the remaining 4 wells.</li>
	</ol>
	</li>
	<li>Place the devices in a bigger plate with an open dish with autoclaved water (wet chamber) and place in the incubator (37 &#176;C, 5% CO2 and 95% humidity) for 1-2 h while preparing the cell culture. See Figure 2b .</li>
</ol>
2. Plating Neurons in Microfluidic Systems
<ol>
	<li>Following the protocol outlined in Ref.8, obtain dissociated hippocampal or cortical neurons from Sprague Dawley rat embryos (either gender).</li>
	<li>Resuspend embryonic neurons in NBM at a concentration of 1-2 million neurons/mL. Verify cell concentrations in the microscope using a hemocytometer and following reference8. Adjust the cell concentration according to the desired cell density. To increase the chances of obtaining single hippocampal axons per channel, plate 10,000 neurons per device. To have multiple axons in the same channel, plate 60,000 neurons per device. 
	Note: These numbers vary according to the neuronal type used.</li>
	<li>Remove the medium from the microfluidic devices without emptying the wells. Leave approximately 5 &#181;L in each.</li>
	<li>To plate cells in the single population device, add 50 &#181;L of NBM to the lower right well. At this point the medium flows by itself to fill the other lower well. Add 20 &#181;L of the concentrate cell solution into the top right well of the microfluidic device, as indicated in Figure 1b .
	<ol>
		<li>To plate cells in the multiple population device add 20 &#181;L of the concentrate cell solution into each of the right wells in Figure 6a.</li>
	</ol>
	</li>
	<li>Check in the microscope if cells are inside the chambers and place the devices in the incubator for 15-30 min to promote cell attachment to the substrate.</li>
	<li>Check in the microscope if there are enough cells in the chambers. If more are needed, repeat steps 2.4 and 2.5.</li>
	<li>Add 50 &#181;L of NBM to the 2 top wells of the single population device and 20 &#181;L of NBM into the same well as the cells were injected in the multiple population device. The media protrudes slightly to form a positive meniscus giving the wells a muffin top aspect. Again, see Figure 2a.</li>
	<li>Maintain the cells at 37 &#176;C, 5% CO2 and 95% humidity.</li>
</ol>
3. Maintaining the Neuronal Cultures
<ol>
	<li>Remove NBM (roughly 30 &#181;L with a pipette) from the cells and apply new pre-warmed NBM the day following their introduction to the devices (that is 1 d after step 2).</li>
	<li>Check every 2 days if there is enough medium in each channel. If the muffin top is low just add more medium to the top wells.</li>
	<li>Culture cells for at least 7 d before removal of the microfluidic devices. The cells can survive in these devices for several weeks. Remove the devices 1-2 days before experiments are performed on samples.</li>
</ol>
4. Removal of Microfluidic Devices
<ol>
	<li>1 - 2 d before removal of microfluidic devices, add 2 mL of NBM prewarmed to 37 &#176;C to each sample dish, flooding the chambers, and maintain the devices in the incubator.</li>
	<li>Use sterile tweezers and one tip to remove the microfluidic devices from the coverslips leaving a patterned configuration of neurons. Use the tip to hold the coverslip in place and the tweezers to clasp the edge of the device at the bottom left corner of the well. Delicately apply torsion, raising the device up with the tweezers so that it peels off the coverslip. See Figure 2c-2d.</li>
	<li>Every 2-3 days, replace half the NBM until the sample is used for experiments.</li>
	<li>Before performing rewiring experiments on the sample, verify that neurites in the single population device channels and the neuronal populations in the multiple population device are isolated by examining the gaps between them in the microscope to ensure there are no filaments linking neuronal populations.</li>
</ol>
5. Preparing PDL-coated Beads
<ol>
	<li>Add 2 x 50 &#181;L drops of either 4, 10 or 20 &#181;m polystyrene beads diluted in water (1:500) to 1 mL of PDL (100 &#181;g/mL). Leave for at least 2 h at room temperature. 
	Note: Protocol can be paused here and resumed the following day.</li>
	<li>Centrifuge the solution at 8,820 x g for 1 min. Carefully remove supernatant without disturbing the beads accumulated at bottom of container.</li>
	<li>Wash the beads twice with 1 mL of sterile 10 mM HEPES pH 8.4 solution.</li>
	<li>Resuspend the PDL-coated beads in 200 mL of 10 mM HEPES pH 8.4 solution.</li>
</ol>
6. Preparing Micropipettes
<ol>
	<li>Prepare pipettes from glass capillary tubes (1 mm inner diameter, 1.5 mm outer diameter) using a horizontal electrode puller. Adjust settings so the outer tip of the pulled micropipette is ~ 2-5 &#181;m. Before pulling, ensure the glass tubes are clean.</li>
	<li>Fix pipettes to glass slides for storage, and ensure that the tip does not contact the surface of the slide as the tip is fragile. Store at room temperature in a covered container to protect from dust. Use pipettes the same day they are pulled.</li>
</ol>
7. PDL-bead Adhesion to Neurons
<ol>
	<li>Add 40-60 &#181;L of PDL-coated beads prepared in step 5 to a cell culture prepared in step 4. Center the pipette tip over the neurons, which are faintly visible on the coverslip, and add the beads (See Figure 3).</li>
	<li>Return the sample to the incubator for 1 h to promote the formation of synaptic contacts8,9.</li>
	<li>After the incubation, remove any un-adhered beads by gently washing the culture with pre-warmed NBM.</li>
</ol>
8. Preparing Physiological Saline Solution (for Room Temperature Experiments)
<ol>
	<li>Prepare physiological saline solution by combining the ingredients listed in references7,8. This is to regulate the cell environment outside the incubator.</li>
	<li>Verify osmolarity and pH levels as indicated in references7,8.</li>
	<li>Continuously infuse solution with O2 to minimize pH fluctuations while conducting experiments.</li>
	<li>Heat to room temperature.</li>
	<li>Set up the perfusion system by inserting one end of a plastic tube (optional dimensions) in O2-infused physiological solution and fixing the other end to a needle inserted in the sample holder. Place the tubing and solution higher than the sample (See Figure 4).
	<ol>
		<li>Disconnect the tube from the needle and connect it to a syringe. Use the syringe to exert pressure and draw liquid, filling the tube. Seal with a roller clamp and reconnect the needle.</li>
	</ol>
	</li>
</ol>
9. Bead Micromanipulation
<ol>
	<li>Install sample in an experimental set-up such that cells can be accessed from above by two micropipettes mounted in micromanipulators and accessed optically below, for instance with the 40X-phase objective (numerical aperture of 0.6) of an inverted optical microscope. In this configuration, mount a CCD camera for image capture on the side port of the microscope. Connect each pipette to 1 mL syringes via plastic tubing. At this step, replace NBM with physiological saline solution (1-2 mL) (See Figure 4).</li>
	<li>During experiments, continuously perfuse cells with the physiological saline solution prepared in step 8 at a rate of 0.5-1 mL/min.</li>
	<li>Select a PDL-bead NOT attached to a neuron in the field of view. Align the bead with a micropipette tip by focusing onto the bead then up to the micropipette. Bring the tip down as close as possible to the bead by monitoring it through the microscope.</li>
	<li>Apply negative pressure with the 1 mL syringe connected to the pipette to pick up the bead. Maintain negative pressure throughout the experiment.</li>
</ol>
10. Pulling Neurites
<ol>
	<li>Select a PDL-bead attached to a neuron in the field of view and attach it to the second micropipette using suction as described in steps 9.3-9.4.</li>
	<li>Pull the PDL-bead-neuron complex by slowly (~0.5 &#181;m/min) moving either the micromanipulator or the sample stage by 1 &#181;m and pause for 5 min to allow neurite initiation.</li>
	<li>Repeat step 10.2 twice. 
	Note: The first 3 &#181;m have to be pulled very slowly to guarantee experimental success, which occurs over 95% of the time.</li>
	<li>Pull the PDL-bead-neuron complex by slowly (~0.5 &#181;m/min) moving either the micromanipulator or the sample stage by 2 &#181;m and pause for 5 min to allow neurite elongation.</li>
	<li>After successful initiation and neurite extension for the first 5 &#181;m, pull the neurite at 20 &#181;m/min over millimeter-scale distances. 
	Note: Pulling can be performed continuously or in steps and at varying rates. See Figure 5b-5c .</li>
</ol>
11. Connecting Neurons
<ol>
	<li>Select a region rich in neurites and lower the PDL-bead-neurite complex so that it physically contacts it. Use other beads to gauge tip height above coverslip surface. See Figure 5d.</li>
	<li>Leave the PDL-bead-neurite complex in contact with the target neurite while manipulating the second micropipette. Lower the second pipette with the second PDL-bead on top of the newly formed neurite about 20 &#181;m form the first bead. Use the second PDL-bead to push the new neurite filament towards the target cell.</li>
	<li>Hold both beads in place for at least 1 h. Verify the absence of focal swelling, a thickening of the neurites contacting the bead, with the microscope16.</li>
	<li>During this time, use perfusion to slowly change the medium of the sample from physiological saline to pre-warmed, CO2-equilibrated NBM.</li>
	<li>Release the bead from the second pipette by releasing suction. If the new neurite remains attached, release the first bead as well. See Figure 5e .</li>
	<li>Gently remove saline solution and replace with NBM (~2 mL).</li>
	<li>Carefully place the sample back in the incubator to strengthen the neuronal connection for future experiments. This connection is stable for &#62;24 h7.</li>
</ol>
12. Verifying the Functionality of the New Connection via Whole-cell Paired Patch Clamp Recordings
<ol>
	<li>Follow references7,18,19. to assemble an electrophysiology set-up.</li>
	<li>Follow references7. to prepare the pre- and postsynaptic electrodes.</li>
	<li>Gather patch clamp data, again following references18,19.</li>
	<li>Compare the results to naturally occurring signals7 to determine the connection type.</li>
</ol>

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The author Margaret H Magdesian is the CEO of Ananda Devices that produces instruments used in this Article.

Using standard micromanipulation and innovative microfluidic devices, a new technique was developed to rapidly initiate, elongate and precisely connect new functional neuronal circuits over large distances. Pipette micromanipulation is a common tool in most neuroscience labs4,13. The real challenge to achieving reproducible and reliable results was standardization of healthy, precisely positioned neuronal cultures for the duration of the experiment (which can be ...

Injuries to the adult central nervous system (CNS) may lead to permanent disability due to multiple mechanisms that limit axonal regrowth1. Following injury, many CNS axons do not form a new growth cone and fail to mount an effective regenerative response2. Furthermore, damage and scar tissue surrounding CNS lesions significantly inhibit axonal growth1,2,3. Current therapies to promote CNS regeneration after injury have focused on enhancing the intrinsic growth potential of the injured neuron and on masking the inhibitors of axonal extension associated with myelin debris and the glial scar1,3. Despite this, the capacity to regenerate long axons to distant targets and to form appropriate functional synapses remains severely limited4,5,6,7.
In the present work, microbeads, pipette micromanipulation, and microfluidic devices are used to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. Previous work has shown that poly-D-lysine-coated beads (PDL-beads) induce membrane adhesion followed by the clustering of synaptic vesicle complexes and the formation of functional presynaptic boutons8. It was also shown that when the PDL-bead is mechanically pulled away after presynaptic differentiation, the synaptic protein cluster follows the bead, initiating a new neurite9. The following procedure exploits this fact along with the ability to culture embryonic hippocampal neurons of rats into organized regions on a coverslip using polydimethylsiloxane (PDMS) microfluidic devices to precisely rewire a neuronal circuit.
These PDMS microfluidic devices are non-toxic, optically transparent and consist of two chambers connected by a system of microchannels. Once assembled on a coverslip, each device serves as a mold to guide neuronal growth and maintain healthy neuronal cultures on precise patterns for longer than 4 weeks in vitro.
Here, a framework is presented in which to investigate the limits of extension and functionality of the new neurite. New, functional neurites are created and positioned to controllably (re)wire neuronal networks. The extension rates achieved are faster than 20 &#181;m/min over millimeter-scale distances and functional connections are established. These results show, unexpectedly, that the intrinsic capacity of these neurites for elongation is much faster than previously thought. This proposed mechanical approach bypasses slow chemical strategies and enables controlled connection to a specific target. This technique opens new avenues for the in vitro study of novel therapies to restore neuronal connectivity after injury. It also enables the manipulation and rewiring of neuronal networks to investigate fundamental aspects of neuronal signal processing and neuronal function in vitro.

<table><tbody><tr><td>Co-culture devices</td><td>Ananda Devices</td><td></td><td>Commercially available at http://www.anandadevices.com</td></tr><tr><td>Neuro devices</td><td>Ananda Devices</td><td></td><td>Commercially available at http://www.anandadevices.com</td></tr><tr><td>No. 1 Glass Coverslip 25 mm Round</td><td>Warner Instruments</td><td>64-0705</td><td></td></tr><tr><td>35 mm Glass Bottom Dishes #0, Uncoated, Gamma-Irradiated</td><td>MatTex Incorporation</td><td>P35G-0-20-C</td><td></td></tr><tr><td>35 mm cell culture dish, Non-Pyrogenic, Sterile</td><td>Corning Inc</td><td>430165</td><td></td></tr><tr><td>95 mm x 15 mm Petri Dish, Slippable Lid, Sterile Polystyrene</td><td>Fisherbrand</td><td>FB0875714G</td><td></td></tr><tr><td>50 mL Centrifuge tubes with printed graduations and flat caps</td><td>VWR</td><td>89039-656</td><td></td></tr><tr><td>15 mL Polypropylene Conical Tube, 17 x 120 mm style, Non Pyrogenic, Sterile</td><td>Falcon</td><td>352097</td><td></td></tr><tr><td>Neurobasal Medium</td><td>Life Technologies</td><td>21103-049</td><td>Extracellular solution</td></tr><tr><td>B-27 Supplement (50X), serum free</td><td>B-27 Supplement (50X), serum free</td><td>17504044</td><td>Extracellular solution</td></tr><tr><td>Pennicilin, Streptomyocin, Glutamine</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>11995-065</td><td>Extracellular solution</td></tr><tr><td>200 &amp;mu; L Pipettors</td><td>VWR</td><td>89079-458</td><td></td></tr><tr><td>2 - 20 &amp;mu;L Pipettors</td><td>Aerosol Resistant Tips</td><td>2149P</td><td></td></tr><tr><td>BD Falcon 3mL Transfer Pipettes [Non-sterile]</td><td>BD Falcon</td><td>357524</td><td></td></tr><tr><td>Glucose</td><td>Gibco</td><td>15023-021</td><td>Extracellular solution</td></tr><tr><td>HEPES</td><td>Sigma</td><td>7365-45-9</td><td>Extracellular solution/Beads</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td>Extracellular solution</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>7447-40-7</td><td>Extracellular solution</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>10043-52-4</td><td>Extracellular solution</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>7786-30-3</td><td>Extracellular solution</td></tr><tr><td>#5 Dumont Dumostar Tweezers 11 cm</td><td>World Precision Instruments</td><td>500233</td><td></td></tr><tr><td>Dissection tools</td><td>Braun, Aesculap</td><td></td><td></td></tr><tr><td>Poly-D-lysine Hydrobromide</td><td>Sigma-Aldrich</td><td>P6407</td><td></td></tr><tr><td>Micro particles based on polystyrene, 10 &amp;mu;m</td><td>Sigma-Aldrich</td><td>72986</td><td></td></tr><tr><td>Borosilicate tubes</td><td>King Precision Glass, Inc.</td><td>14696-2</td><td></td></tr><tr><td>Horizontal Pipette Puller</td><td>Sutter Instruments</td><td>Brown-Flaming P-97</td><td></td></tr><tr><td>Micromanipulators, PCS-5000 Series</td><td>SD Instruments</td><td>MC7600R</td><td></td></tr><tr><td>1 mL Syringe</td><td>BD Luer-Lok</td><td>309628</td><td></td></tr><tr><td>Inverted Microscope</td><td>Olympus&amp;nbsp;</td><td>IX71</td><td></td></tr><tr><td>Objective</td><td>Olympus</td><td>UIS2, LUCPLFLN 40X</td><td></td></tr><tr><td>CCD Camera</td><td>Photometrics</td><td>Cascade II: 512</td><td></td></tr><tr><td>Leibovitz&#039;s (1x) L-15 Medium</td><td>Life Technologies</td><td>11415-064</td><td>Rat Dissection</td></tr><tr><td>Typsin-EDTA (0.05%), Phenol red</td><td>Life Technologies</td><td>25300054</td><td>Rat Dissection</td></tr><tr><td>DMEM (1x) Dulbecco&#039;s Modified Eagle Medium [+4.5 g/L D-Glucose, + L-Glutamine, + 110 mg/L Sodium Pyruvate]</td><td>Life Technologies</td><td>11995-065</td><td>Rat Dissection</td></tr><tr><td>HBSS (1x) Hank&#039;s Balanced Salt Solution [- Calcium Chloride, - Magnesium Chloride, - Magnesium Sulfate]</td><td>Life Technologies</td><td>14170-112</td><td>Rat Dissection</td></tr></tbody></table>

rewiring neuronal circuits: a new method for fast neurite extension and functional neuronal connection

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

Embryonic rat hippocampal neurons are cultured in microfluidic devices to enable precise positioning of cells, PDL-beads and micromanipulators. The first step is to properly assemble the microfluidic device on a glass coverslip or dish. It is essential that the microfluidic device be well attached to the substrate to avoid cells exiting the chambers and moving under the parts of the device that should be sealed (Figure 1a). To maintain healthy cultures for se...

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

The overall goal of this procedure is to initiate new neurites, rapidly extend them, and precisely connect them to target cells. This procedure is only possible by using cells grown in a controlled and reproducible environment. This method allows us to answer key questions in neuroscience.What limits the growth rate of neurons? How fast can they grow? Do mechanical clues play a role?We can also study how synapses are formed, how do neurons find a target cell? How do synapses age, mature? And thirdly, we can start building systematically neural networks, study how neurons compute.The main advantages of this technique are that it enables easy manipulation, experimental reproducibility, and ultimately a high degree of control at the single-cell level. To begin this procedure, clean and prepare the desired number of sterile cover slips. Next, coat each cover slip with 0.5 to 1 milliliters of 100 microgram per milliliter PDL for at least two hours at room temperature.After that, wash the cover slips twice with water and remove all the liquid. Let them dry in a sterile environment for 5 to 10 minutes or until the surface is completely dry. Then, place the microfluidic devices with the patterns facing up under the UV light in a biosafety cabinet for 10 minutes.After 10 minutes, place a microfluidic device with the pattern facing down in contact with the clean cover slip. Use the tweezers to softly press the device so it adheres to the glass. To fill the single population device with medium, add 50 microliters of complete cell medium to the right upper well.And then add another 50 microliters of complete cell medium to the well diagonal to it. To fill the multiple populations device with medium, add 30 microliters of complete NBM to one row of wells. Make sure the medium flows between the wells.Then, add 50 microliters of medium to each of the remaining wells. Place the devices in a bigger plate with an open dish of autoclaved water, and place the dish in the incubator for one to two hours while preparing the cell culture. Now, remove the medium from the upper wells of the microfluidic devices without emptying the wells.Then, add 20 microliters of the concentrated cell solution into the top right well of the microfluidic device, and check in the microscope how cells flow inside the single population microfluidic device. To plate cells in the multiple populations device, add 20 microliters of the concentrated cell solution into each of the top wells. Under the microscope, check if the cells are inside the chambers.Afterwards, place the devices in the incubator for 15 to 30 minutes to promote cell attachment to the substrate. After the cells attach to the substrate, add 40 microliters of NBM to the two top wells of the single population device. This will help remove the air bubbles from the micro channels.Add 20 microliters of MBM to the top wells of the multiple populations device, the same wells where the cells were injected. The media should protrude slightly to form a positive meniscus, and give the wells a muffin top aspect. One to two days before the microfluidic device's removal, add two milliliters of pre-warmed NBM to each sample dish and flood the chambers.Then, keep the devices in the incubator. Subsequently, use sterile tweezers and a pipette tip to remove the microfluidic devices from the cover slips leaving a patterned configuration of neurons. In this procedure, and 40 to 60 microliters of the prepared PDL coated beads to the cell culture.Then, return the sample to the incubator for one hour to promote the formation of synaptic contacts. Subsequently, install the sample in an experimental setup such that the cells can be assessed from above by two micropipettes, and also be accessed optically. In this configuration, a CCD camera is located on the side port of the microscope for image capture.Connect each pipette filled with saline to a one milliliter syringe, then perfuse the sample with physiological saline solution. Next, under the microscope, check the gap between the two isolated neuronal populations to insure that the two populations are not naturally connected. Select a PDL bead that is not attached to the neuron in the field of view.Align the bead with a micropipette tip by focusing on the bead and then on the micropipette. Subsequently, bring the tip as close as possible to the bead by monitoring it through the microscope. Then, apply negative pressure to the pipette to pick up the bead, and maintain the negative pressure throughout the experiment.Now, select a PDL bead attached to a neuron in the field of view, and attach it to the second micropipette using suction. Pull the PDL bead neuron complex by slowly moving either the micromanipulator or the sample stage at 0.5 micrometers per minute for 1 micrometer, and pause for five minutes to allow neurite initiation. Pull the PDL bead neuron complex by slowly moving it laterally at 0.5 micrometers per minute over two micrometers, then pause for five minutes to allow neurite elongation.After successful initiation and neurite extension for the first five micrometers, continuously pull the neurite at 20 micrometers per minute. To connect the neurons, select a region rich in neurites. Use other beads to gauge the pipette tip height above the cover slip, and lower the PDL bead neurite complex so that it physically contacts the neurites.Leave the PDL bead neurite complex in contact with the target neurite while manipulating the second micropipette. Then, lower the second pipette with the second PDL bead on top of the newly formed neurite about 20 micrometers from the first bead, and use the second PDL bead to push the new neurite filament towards the target cell. Next, hold both beads in place for at least one hour.Verify the absence of focal swelling, a thickening of the neurites contacting the bead, with the microscope. During this time, slowly change the medium of the sample from physiological saline to pre-warmed carbon dioxide equilibrated NBM. Release the bead from the second pipette by releasing the suction.If the new neurite remains attached, release the first bead as well. Afterward, carefully place the sample back in the incubator to strengthen the neuronal connection for future experiments. You can use whole cell patch clamp recordings to investigate the electrical functionality of the micromanipulated connection.Shown here are the isolated neuronal populations separated by a 100 micrometer gap in PDMS microdevices. Pair to patch clamp recordings were performed in whole-cell configuration, and the mechanically induced connections were analyzed, and compared with those in naturally connected neuronal populations, and non-connected populations. The electrical responses after pre-synaptic action potential recorded from the neurons connected naturally and by micromanipulation are significantly higher and temporarily correlate with the pre-synaptic activity.Once mastered, this technique can be performed in eight hours if done properly, and while attempting this procedure, it is important to remember to have healthy and well-patterned long term cell cultures to guarantee fast and reproducible results. Following this procedure different methods can be used to verify the electrical functionality of the micromanipulated connection. After watching this video, you should have a good understanding of how to grow cell populations in microfluidic chambers, and use pipette manipulations to initiate, extend, and connect neurites.

rewiring-neuronal-circuits-new-method-for-fast-neurite-extension

Research

JoVE Journal

Neuroscience

Perspectives on Neuroscience

This video demonstrates a technique for the rapid initiation and extension of a neuron and creation of new connections with a target neuron using a microfluidic device and micromanipulation. This could be used for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

Initiating Rapid Neurite Extension and Formation Functional Neuronal Connections

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human induced Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered "living scaffolds", exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Neurite outgrowth assays provide a quantitative value about regenerative neuronal processes. The advantage of this semi-automatic software is that it segments cell bodies and neurites separately by creating a mask and measures various parameters such as neurite length, number of branch points, cell-body cluster area, and number of cell clusters.

Standardization of a Novel Semi-Automatic Software for Neurite Outgrowth Measurement

Learn to rapidly initiate, extend and connect neurites using poly-D-lysine-coated beads and micropipette guidance in microfluidic chambers. This method enables precise control of neuronal growth for neuroscience research.

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

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